1. Field of the Invention
In a parent application and a first continuation-in-part application thereof, discovery of a broad class of dyestuffs were disclosed which, under visible light range absorbance in a supravital blood analysis, provided notable advantage in the field of cytology.
This application is a continuation-in-part of U.S. Ser. 242,662 filed Mar. 11, 1981, and is related to the same subject matter as the first above Parent case which provides an improved method for optical differentiation of the five individual white blood cell species by use of a class of basic quaternary metachromatic dyes which were found to supravitally stain each of the species within a temperature range.
The subject matter of the first continuation-in-part application relates basically to a similar area of determination but is founded on the discovery that certain specific sub-classes of the dyestuffs broadly useful for differentiation, identification and enumeration of human blood cell leukocytes were also singularly useful as well in differential determination of developmental stages of neutrophilic granulocytic cells. The foregoing inventions were reduced to practice using light waves of the same wave length as present in ordinary daylight herein sometimes call white light spectrum.
Since reduction to practice of the first continuation-in-part application, U.S. Ser. 242,662 of Mar. 11, 1981, microscopic examinations of a supravitally dyed field has been made available which permits the observer to practice the general method of supravital human blood analyses as originally disclosed using "white" light (an electromagnetic energy form of radiation having a wave length of from about 4000 to 7700 angstroms), but now enlarged to include fluorescent light emissions. In the Parent and the Continuations white light spectra passing through the supravitally dyed specimen field is absorbed. Fluorescent light is related to light emissions and may be caused by the flow of some form of energy into the emitting body. Emission spectra of fluorescence results from the flow of energy being absorbed and light being emitted in characteristic frequency.
Fluorescent light emission for the purposes herein in the fluorescent microscope may be ultra-violet, violet and sometimes blue radiations. Mercury vapor light is commonly used. However, early experimentation points to the value of a single, coherent beam of light, as from laser technology, to be of use as well in some instances. It is known that in flow cytometry apparatus laser beams have been used to excite single cells exposed to acridine orange dye in cell sorters. Instruments can look at a single cell, determine its specific patterns and measure the intensity of the fluorescent colors. However, acridine orange has serious limitations being both pH and temperature dependant. Acridine orange does not exhibit useful metachromasia under an absorbtive light mode. Additionally, the dye tends to diffuse out and is not useful in supravital examination of (living) cells, e.g., it is concentration dependant. In the presently disclosed methods, laser light stimulation of prepared microscopic cells dyed with basic orange #21, for example, provides unique means for identification, differentiation and enumeration of blood cells and other tissues capable of manufacturing, transferring or storing blood cells. Laser light does not appear to excite or bring out a new quality of chromasia or interfere with metachromasia, so important a factor in the use of the dyestuffs of this invention. It can be focused with high power density within one cell making them more distinct and the measurement more precise. The colors emitted appear to be unchanged by the form of energy causing the sorbed dyes of this invention to fluoresce.
Using the specific groups of dyes herein disclosed and claimed, but particularly basic orange #21 which is unique with fluorescent means, and also as hereinbefore disclosed in the Parent case and the first Continuation-in-part also unique with white light absorbance, the art of cytology is immeasurably advanced.
Dyes originally disclosed in the Parent application have been broadly classed as methines, polymethines and cyanine dyes. Dyes broadly within the class include carbocyanines, merocyanines, azacyanines, oxanols, etc. However, so very few of this broad class have been found to be metachromatic and useful, particularly in the present field of use where they must also be metachromatic under fluorescent conditions as well as white light absorbance.
2. Description of the Prior Art
Ehrlich made biological elements more readily and easily recognized under microscopic examination and for photographic observation by use of dye stains (aniline dyes) to identify certain white blood cells. Ehrlich was the first to note that some dyes were metachromatic, observing that the staining of the cell or components such as granules of leukocytes causes the cell to take on a color different than that of the stain in solution or expected color from the stain. Basophils, for example, were observed to take on a color different from the stain. Other histological specimens other than blood cells have also been reported to stain in a plurality of identifiably different colors.
A review of the state of the art indicates it is almost universal practice, before staining (which presently uses a plurality of chemically differing dyestuffs in admixture) to employ a fixative procedure which may require up to an half hour treatment before the biological specimen is subjected to dye stain. Fixatives are generally preservatives and denaturants that often interfere with the sensitivity of the dye sorption. Illustratively, fixatives include formaldehyde both as liquid and vapor, absolute alcohols (methyl), picroformal, etc. Very often living cells do not stain using vital dyes and fixatives have been essential to staining the specimens. Cytochemistry includes considerable information on techniques developed to assure reproducible staining of blood cells. Many essential additives are normally unstable and deteriorate rapidly, thus making cellular identification difficult and in some instances unreliable. Dr. Thomas E. Necheles has observed in relation to leukocyte analysis that this "system has undergone little or no change in fifty years."
Dye staining does serve, however, as a means of discernment of otherwise undiscernable detail of conferring a color reaction on cells and their stainable components; metabolic, functional or pathological.
United States hospitals began leukocyte counting in the early 1900's, using the count as indicia as to whether emergency surgery was necessary, for example. In the U.S. alone, more than half a million differential counts are performed every day, most of them by manual methods. It is important that total white cell counts and differential cell counts be performed and reported without delay. Time is of essence and providing required analysis more rapidly is a desideratum.
The value of leukocyte counting having been established, the demand for rapid blood analysis has developed so that beginning about 1950 with the work of Mellors and Papincolaou (1952) development of automated differential leukocyte counting instrumentation means had developed into a plurality of intruments by 1980. The CYDAK unit was early used to investigate the feasability of blood cell classification which pointed up the importance of specialized staining procedures and features were extracted from optical density histograms of each cell image. The procedure established that cells could be differentiated into four of the five classes of leukocytes, namely; neutrophils, eosinophils, lymphocytes and monocytes. Young (1969) published results on an automated classification of five cell classes and Bacus in 1971 extended the differentiation.
However, it is understood that automated differential systems presently rely upon multiple dye usage and dye degradation systems or indirect fluorescent measurement using fluorescent dyes.
In the prior art staining of blood it has been observed that it is practice to use two or more stains in combination (Romanowski, Giemsa and Wright stains). These methods are difficult in practice to provide quality control. The methods require standardization in preparation of each dye stain component as well as in the method of specimen staining. In development of successful automated leukocyte counters, reproducibility of staining is even more important to verifiable analysis.
LARC stainer (used in commercial automated differential leukocyte counter) is reported (Mogler 1973) to be a mixture of some ten thiazine dyes, oesin Y and 2.sup.1, 4.sup.1, 5.sup.1 tribromofluorescein (P. N. Marshall). Present art stains most often are in fixative alcoholic solutions and employ two or more stains in combination. Accurate analysis of vital blood staining is made most difficult. With the difficulty presented in the controlled oxidation of methylene blue essential to Romanowski stains, for example, the problems of quality control of the added ten individually different dye stains as are used in combination become awesome.
It has been recognized in the art that the widespread standardization and adoption of a limited number of stains would ensure greater accuracy and reproducibility in cytological studies. Serious introduction of artifacts have been observed by use of fixatives and cause difficulty in interpretation and misinterpretation and leukocyte differentiation and enumeration. pH adjustments, heavy metal cations have been reported to prevent cytochemical tests from working in the expected manner. Some dyes, particularly azo dyes, are noted to demonstrate non-specific precipitation around cells; other degenerative changes in fixed blood samples include vacuoles, clover-leafing of nuclei, distortion cell shapes and smudges and interference with ideal staining. The importance of performing differential counts on as near living cells in the shortest possible time in order to obtain optimally useful and valuable blood cell analyses has been recognized. Alcoholic dye solutions interfere with supravital staining. So far as is known, freshly prepared water soluble stains exhibit a minimum denaturant effect upon supravital blood during examination. All dyestuffs are more or less toxic to the blood cells, but some are more so that others. It is material that the cells under examination remain living as long as possible. Rapidity of staining obviously shortens the exposure time, thus allowing greater opportunity to examine leukocyte cells before all vitality is lost. Automated differential leukocyte counting in less minutes is sought for.
Studies and review of the prior art of performing microscopic blood analyses and disease diagnosis has indicated it is not unusual for pathologists to warm the dye and the blood specimen to body temperatures (about 37.degree. C.) before contact. Dr. Sabin had a "warm box" to insure temperature control.
It has also been noted that some dyes used in the prior art are quite temperature sensitive. The literature reports that cresylecht violet is not an operative stain above 30.degree. C. It is considered important for the purposes of this method as disclosed herein that the dyestuff be useful to stain leukocytes at temperatures as high as 37.degree. C. and no difficulty has been observed with the select dyes to temperatures of about 40.degree. C.
In the Parent application, a relatively small number of metachromatic dyestuffs are disclosed as useful in identification of one or more species of leukocyte. Identification and differentiation was specifically related to polymophonuclear leukocytes (neutrophils), eosinophils, basophils, lymphocytes generally, and monocytes. A uniting commonality observed was that all of the dyes found to be operative for the purposes of the Parent application metachromatically stained monocytes differentially from others in the above group.
The unusual qualities of the dye basic orange #21 (CI #48035 and Spectral Curve 7) were observed in relation to the eosinophils, basophils, and monocytes, but as the B-cells are few in number they were initially overlooked. It was initially observed in the Parent case that optical differentiation between mature and immature neutrophils appeared potential in that the mature granules were different in chroma from the immature granules which were more red and orange in comparison. As this group, including myeloblasts, promyelocytes, myelocytes, metamyelocytes and bands are not always present in all blood specimens or present in significant numbers as is often the case with T-lymphocytes (or T-cells) and B-lymphocytes (or B-cells) they were not then all specifically identified as being metachromatically and differentially stained by basic orange #21.
Subsequent to completion of the work supportive of the Parent application, continuing research on the use of this unique dye in similar blood donor studies established that it was reproducibly possible, using this selected basic cationic dye of the methine, polymethine and quinoline class to distinguish through metachromatic response certain lymphocytes. It is also possible further to identify at least ten recognized granulocytes and lymphocytic cells established in the art to be of vital interest to the health sciences Platelets, also identified as thrombocytes, can also be identified and enumerated.
Further, this differentiation was immediate, it required no complex biochemistry or arduous pre-treatment of the blood specimens. Additionally, it was noted the dye exhibited minimum toxicity.
Micro spectrophotometric measurements were made with an aperture small enough to measure the color in the granules of supravitally stained leukocyte granulocytic cells. No other part of the cell entered into the measurements to any extent were found to provide extinction coefficients of the colors of the different leukocyte species which were consistently different and were often of an order of differences in hue, value or chroma of the order of 50 nanometers. These were recognizable peaks, consistent over many cells. It is understood that differences of the order of 5 nm are significant in microspectrophotometric measurements if the differences are consistent and reproducible.
Among the immature granulocytic cells immediately identifiable and distinguishable one from the other are myeloblasts and cells of the myeloid series, namely; promyelocytes, myelocytes and metamyelocytes. These are believed to be and are generally understood to be precursors of the polymorphonuclear leukocytes or neutrophils, which are also stained metachromatically so as to be readily and easily distinguished, identified and enumerated by the supravital blood analyses made possible by the advances disclosed herein.
As disclosed in the parent application, it is also practical at the same time to distinguish neutrophils, eosinophils, basophils, lymphocytes and monocytes from each other and from the foregoing precursors should they all be present in a specific blood sample under microspectrophotometric analyses.
Additionally, it has also been found that this unique dye provides an optically different pattern of color as well as a different density of each color of granule in bands, neutrophils and leukocytes. Thus, this quality of leukocyte cell can also be uniquely separated by optical differentiation from the other immature cells identified above. The differentiations in color, color arrangement and color density are also of such a degree of magnitude of difference that both human counting manually of all the above individually named cells can be accomplished, both with white light spectra (absorption) and fluorescence (emission).
Sources of fluorescent emission energies are, for example; mercury vapor lights, tungsten-halogen sources, lasers, deuterium sources, zenon, etc.
Evidence available also indicates automatic differential counting equipment will develop based upon and to be accommodated by differences due to the presence or absence of color and the physical patterns established in the nucleus and by the relative number, size, arrangement or pattern and hue, value and chroma (color) and color density due to the number of granules in the cytoplasm. The duality of colors under bimodal light exposures provides a double check on observation and a means of discovery of differentials in cell structure.
Almost unbelievably, but also demonstrated in the basic research thus far completed, is the further ability to differentiate B-lymphocytes or B-cells from T-lymphocytes or T-cells. Again, it is possible to spectrally identify each of these important lymphocytes, one from the other, qualitatively and quantitatively using the same dyestuff in the same supravital, fixative free analysis as well as to distinguish and enumerate immature and mature cells including bands. T-lymphocytes have been observed and identified in lymph node tissues and other tissues associated with white blood cell metabolism by means of the fluorescent mode of observation.
Earlier discovery of the capacity of basic organe #21 to differentiate, in addition to those cells disclosed in the Parent application, myeloblasts and blood cells of the myeloid series as well as bands and T-lymphocytes and B-lymphocytes extends the original potential field of usefulness of the dye unexpectedly beyond the capacity recognized in the Parent application. Supravital blood specimen fractions of fluids associated with healthy tissue or tissue suspected of abnormality such as plasma, lymph, serum, etc., containing one or more of the above cells after metachromatic staining may be examined microscopically under this bi-modal energy system herein described, or by use of either light source alone and thus differentiate each species of cell indicated above permitting enumeration and comparative study.
The present advance in the art, coupled with the parent disclosure establishes unparalleled advance in hematology, cytology and immunology and the ability to plan and conduct researches in an unlimited area of human health. Need for costly reactants, invaluable research time and more accurate data assembly have been thereby measurably advanced.
The art of diagnosis of disease has a new horizon beyond the present limits with the finding of fluorescent responses using a limited few of the basic quaternary cationic chemical class that has produced the unique dyes of this disclosure.
Initial observations made frist in the parent application (U.S. Ser. 129,680, filed Mar. 12, 1980) using a Zeiss fluorescent microscope revealed that carbocyanine K-5, a methine-polymethine class of dye, to be metachromatic both under white light absorbance and emitted fluorescence. Later examination of a large group of dyes within the above chromophore classification including reds and violets (listed below) and basic orange 21 all were found to supravitally dye monocytes to exhibit this dually metachromatic or bi-modal light response under the Zeiss fluorescent microscope.
Pursuing observation that basic orange #21 had shown not only metachromasia under white light spectra, but also under conditions of fluorescence stimulated continued research establishing that basic orange #21 is operable under fluorescent light sources to produce substantially the same patterns of cell geometry and arrangement in the same cells as were disclosed in the Continuation-in-part identification above. However, the fluorescent colors were not of the same color response as with white light sources, though the geometric patterns were entirely corraborated. Parallel examinations of the same prepared slide in a large number of instances of both normal leukocytes and those of patients with various stages of diseased conditions both under normal white light wave lengths, as was the subject matter of the parent application (U.S. Ser. No. 129,680), and under fluorescent light, as here, produced a remarkable demonstration of repeated leukocyte identifications and confirmations under both white light wave lengths and under fluorescent light wave lengths but with identifying colors of both different in bi-modal means of observation in hue, value and chroma and of varying visible light intensity, as well.
Studies carried forward using species of methine and polymethine dyes in leukocyte cell identifications continued to confirm unusual properties of certain dyes in this class, particularly basic orange #21, basic red #13, basic red #36, basic red #49, basic violet #7,15, #16, #36, #39 and #40. All of the just identified dyes were found to be metachromatic under white light wave lengths and under fluorescent emission and all instantly stained monocytes characteristically and metachromatically under the bi-modal means here described. Further tests determined that all of the foregoing dyestuffs were quite unusual in that they are also metachromatic in conjunction with certain biological specimens under fluorescent light and were also metachromatically fluorescent when used as supravital to stain monocytes.
Samples of dyes located in a world-wide search total about 2,000 in number and have been subjected to testing. Many of these dyes are no longer available from or through known dye sources.
Specific studies of basic orange #21 further reveal it to be unique among the dyes numbered above. Basic orange #21 is the only dye presently known which exhibits a bi-modal function for identification of all the biological blood cells named below. This unique dye functions under both white light spectrum and fluorescent light spectrum stimuli in a metachromatically different identification of each individual ones of the following leukocytes including the developmental stages of neutrophilic, granulocytic cells. Rapid supravital staining with aqueous solutions of basic orange #21 makes possible optical differentiation, identification and enumeration under fluorescent light spectra, for each one of the following cells; that is, with one prepared microscopic field and with either manual, sequential or simultaneous examination stereo-optical devices under white light spectra and fluorescent spectra stimulus. Two different but characteristically distinctive color patterns become available and each can be checked against the other to confirm identification of neutrophils, eosinophils, basophils, monocytes, lymphocytes, promyelocytes, myelocytes, metamyelocytes, bands and B-cells as well as T-cells! Time has not permitted exhaustive study of possible limitations of more advanced computer operated high technology devices where simultaneous readings of bi-ocular screens image both white and fluorescent light projections from a single specimen field as possible.
Optical devices are known which permit both simultaneous and sequential bi-modal analyses of apparatus useful for simultaneous measurement of absorption and fluorescence. (See page 144, J. Membrane Biology 33, 141-183 [1977].COPYRGT.. Springer-Verlag, New York, Inc., 1977). Thus it is not unknown to subject a dyed biological specimen to observation under the stimulus of a bi-modal light source.
It is further a matter of record that fluorescence alone is often an easier, faster and more versatile light source in stimulating microscopic differentiation, but it is also known that this light source may introduce complications in calibration and accuracy due to artifact vulnerability. However, by making possible sequential use of both white light wave length and fluorescent light wave length in a comparative observation of a single biological test specimen dyed with a dyestuff that is not only metachromatic under white light spectra but metachromatic under fluorescent light spectra makes possible observation of both similarities and differences existant in the characteristic known components of leukocytes as to their nuclei primary granules, secondary granules, etc. Unique aspects of cell structure of eosinophils have been observed which assist in their differentiation, identification and enumeration.
The granules of eosinophils specifically show strong fluorescence only around the periphery of the granule in a "case" or "shell" pattern. As far as ascertained, this border or peripheral fluorescent pattern or shell uniquely identifies eosinophils. Such a structural indicia of a blood cell has not heretofor been known to have been described. The observation suggests that white blood cells are releated tissues upon further comparative studies under both fluorescence and white light spectra will be found to reveal further avenues of discovery and stimulate novel studies through newly observed apparent structural differentiation not even suspected to exist heretofore.
Another illustration of promise is a noted difference in the degree of intensity of nuclear fluorescence among various T-cells (T-lymphocytes). It is anticipated that these observed differences in fluorescent light emission provide clues to identification of T-cell sub-sets, e.g. supressor and killer cells. While it is recognized that there is an observable significant difference in T-cells under bi-modal light illumination, the significance of the differences noted is not presently understood.
Present vital interest in immune studies suggest a locus of practical interest and application of the bi-modal observations possible with the supravital analytical methods hereby introduced. Observations of leukocytes and their developmental stages and the recognition of structural differences of the biochemistry and biophysics through bimodal light observations will lead to deeper understanding of their order and the disorders of disease.
A study of the chemical structures of basic orange #21 and base orange #22 was conducted upon finding the first to be most unusual and the second inoperative for the purposes herein.
The only differences to be observed are that the indolyl radical of each basic orange varies only by a change in the methyl group from a 2 position in basic orange #21 to a 1 position in basic orange #22. The methyl group is substituted on a carbon in basic group #21 and on a nitrogen group in basic orange 22. The 2 position in basic orange #22 has a phenyl group in place of the methyl group of basic orange #21 at the 2 position in the structure.
Prior art references indicate that it was not unusual in supravital analyses to employ three concentrations of dye in three preparations of slides in such analyses as are an essential check on results. With basic orange #21, the color differentials are so separated and the colors so exceptionally vivid that one can readily distinguish primary from secondary granules, instantly, with one dye and one slide, and with either white light spectra or fluorescent light spectra.